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Khajuria et al. J Transl Genet Genom 2020;4:91-103 I http://dx.doi.org/10.20517/jtgg.2020.06 Page 101

the findings of previous studies [3,7,8,16] . All variations identified in the patients were de novo. In one of the
families, the mother was found carrying a different novel variant than her daughter and has no symptoms
[25]
of the disease, as we reported previously .
Bioinformatic analysis revealed that most of the MECP2 missense variants clustered in MBD of MeCP2
were of damaging or deleterious nature, whereas all the missense variants identified in CTR were predicted
as benign or non-deleterious. These findings support previously reported studies on RTT patients [26,27] .
Only two missense variants were identified in TRD of MECP2 and bioinformatic analysis of the recurrent
missense variant p.R306C in TRD predicted it as damaging, whereas the other non-recurrent missense
variant p.T228S was predicted as benign or non-deleterious. The patient carrying this variant p.T228S was
carrying another deleterious variant, p.P152R. As the number of missense variants identified in TRD in the
present study was small, the effect of these variants could not be explored, but the findings of the present
study strongly indicate that sequence variations in MECP2 gene are the major cause of classical RTT.

There are many studies on genotype–phenotype correlation in RTT patients from 2001 to 2016 but the
results are inconsistent [16,18,21,27-32] . This inconsistency can be due to the use of different diagnostic criteria
and severity scales for evaluation of the patients. There are currently no data on genotype-phenotype
correlation in Indian patients with RTT. In this study, we tried to correlate the type and position of
identified sequence variants with the phenotype of the patients.

When different types of sequence variations were compared with the phenotype, it was found that patients
carrying early truncating variants showed more severe phenotype as compared to the patients with late
[18]
truncating and missense sequence variants, supporting the findings from a previous study .
While comparing the types of sequence variations with their location in MECP2, it was found that the
variants leading to severe phenotype were clustered more in functional domains of MeCP2. Only 5% of
early truncating variants were present in MBD and CTR as compared to 20% present in the TRD. Only
5% of late truncating variants were observed in the CTR of MeCP2. The rare presence of missense variants
in TRD or CTR of MeCP2 as compared to MBD can be explained on the basis that missense sequence
variations may have mild impact on protein function compared to truncating sequence variations, resulting
[26]
in a mild phenotype. These findings are in support of a previous study . The only recurrent missense
variant in TRD was p.R306C and the other missense sequence variations found in TRD and CTR (p.T228S,
p.E394K, p.E397K, and p.P430S) were observed with single occurrence, supporting the previous hypothesis
[26]
that most of the missense sequence variations within the TRD might be benign variants .

In conclusion, this study presents the largest cohort describing the molecular genetics of classical RTT from
India. To the best of our knowledge, this is the first study showing the highest detection rate of MECP2
variants in the patients with classical RTT and supports that clinical stringency based on revised diagnostic
criteria can increase the variant detection rate. We propose the following MECP2 screening strategy in
Indian patients with Classical RTT. Exon 3 of MECP2 should be screened first, followed by exons 2 and 4
using Sanger sequencing, and, in turn, followed by quantitative analysis using MLPA. The present study
adds information on the molecular characterization of Indian patients with RTT and also reports 13 novel
variants expanding the genotypic spectrum of RTT. The findings can be useful for diagnostic testing,
genetic counseling, and prenatal testing.

Limitations and future research
Although the study was performed on a large cohort of patients, we were unable to prove the functional
impact of novel variations on the MECP2 protein as only software prediction tools were used. It would
be useful to conduct functional studies for the new variants identified. A review of the current literature